Target Discovery and Validation Reviews and Protocols

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In Vivo Assay of Human Angiogenesis 261 13. PDGF-β LightCycler kit (Search-LC). 14. Human PDGFB-specific PCR primers 5′-CAT AGA CCG CAC CAA CGC CAA CTT C-3′ as forward primer and 5′-ATC TTT CTC ACC TGG ACA GGT CGC AG-3′ as reverse primer. 15. Human hydroxymethylbilane synthase-specific PCR primers 5′-GTA ACG GCA ATG CGG CTG-3′ as forward primer and 5′-GGT ACG AGG CTT TCA ATG-3′ as reverse primer. 16. QuantiTect SYBR Green PCR kit (QIAGEN GmbH). 17. LightCycler (Roche Diagnostics). 2.5. In Situ Hybridization 2.5.1. Probe Generation and Labeling 1. A chloramphenicol-resistant pBC Vector containing a 1-kb insert of hPDGFB was kindly provided by C. Betsholtz (Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden). 2. EcoRI and NotI restriction enzymes (New England Biolabs). 3. Phenol/chloroform extraction: phenol saturated TE8, phenol isoamyl alcohol, sodium acetate, 96% ethanol, and 70% ethanol. 4. DIG RNA labeling kit (Roche Diagnostics). 5. T7 and T3 RNA polymerases (Roche Diagnotics). 6. Ribohybe (Ventana Medical Systems). 2.5.2. In Situ Hybridization 1. Ventana Discovery automated histology robot (Ventana Medical Systems). 2. Ventana solutions as supplied by Ventana Medical Systems: mild alkaline protease 3, Ribo CC, standard saline citrate (SSC), and BlueMap kit for developing the nitroblue tetrazolium salt/5-bromo-4-chloro-3-indolyl phosphate alkaline phosphatase substrate. 3. Biotinylated anti-digoxin antibody (Jackson ImmunoResearch Laboratories). 4. DIG-labeled RNA probe. 2.5.3. Double Staining of In Situ Hybridized Sections With Ulex Lectin 1. 70, 96, and 100% ethanol baths. 2. Xylene bath. 3. FITC-labeled Ulex lectin and polyvinyl alcohol (PVA). 3. Methods 3.1. Cell Preparation 1. Isolate HUVECs from the umbilical vein with 0.2% collagenase as described by Jaffe et al. (32). 2. Culture HUVECs in complete MCDB-131 (containing 7.5% fetal calf serum, 10 ng/mL rhEGF, 1 ng/mL rhbFGF, 1 µg/mL hydrocortisone, 50 µg/mL gentamicin,
262 Skovseth, Küchler, and Haraldsen Fig. 2. Animal handling. (A–C) Injection of Matrigel. (D–F) Implantation of an endostatin-filled microosmotic pump. (G) Harvesting of a Matrigel plug for RNA analysis. (H) Vascularized Matrigel plug. Note the Matrigel plug after injection in C and the polymerized Matrigel under the skin in G (both marked with a circle).
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METHODS IN MOLECULAR BIOLOGY 360 T
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M E T H O D S I N M O L E C U L A R
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© 2007 Humana Press Inc. 999 River
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vi Preface get. Approaches to targe
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viii Contents 10 Transgenic Animal
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x Contributors SAHOHIME MATSUMOTO
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xii Contents of Volume 2 12 Validat
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2 Sioud disease pathogenesis and pe
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4 Sioud A more fruitful approach fo
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6 Sioud both transient and permanen
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8 Sioud serum biomarkers. This syne
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10 Sioud Fig. 2. Schematic of targe
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12 Sioud 13. Magin, T. M. (1998) Le
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Harnessing the Human Genome 15 The
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Harnessing the Human Genome 17 Fig.
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Harnessing the Human Genome 23 The
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Harnessing the Human Genome 27 repr
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Harnessing the Human Genome 29 14.
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Harnessing the Human Genome 31 45.
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34 Imoto et al. (4,5), and Bayesian
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36 Imoto et al. 2. Materials Two ty
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38 Imoto et al. Fig. 3. Graphical v
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40 Imoto et al. Fig. 5. Result of t
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42 Imoto et al. p(D |G) = ∫ p(D,
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44 Imoto et al. β k (k = 1,…,q j
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46 Imoto et al. Fig. 7. Partial res
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48 Imoto et al. Fig. 8. Strategy to
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50 Imoto et al. Fig. 9. (continued)
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52 Imoto et al. Table 3 List of Roo
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54 Imoto et al. 5. De Hoon, M. J. L
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56 Imoto et al. 39. Kamimura, T., S
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58 Beaty et al. cytotoxic drugs and
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60 Beaty et al. Fig. 1. Principles
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62 Beaty et al. oligonucleotide mic
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64 Beaty et al. 3. The 12% polyacry
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66 Beaty et al. 2.3.3. Protein Stai
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68 Beaty et al. 3.1.3. Labeling of
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70 Beaty et al. 1 µL of the anneal
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72 Beaty et al. 4. Phenol:cholorfor
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74 Beaty et al. 19. Wash twice with
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76 Beaty et al. The Following Volum
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78 Beaty et al. using a protein ass
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80 Beaty et al. 6. Shake the gel fo
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82 Beaty et al. Fig. 2. Preparation
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84 Beaty et al. search in. A widely
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86 Beaty et al. 3. Kern, S. E., Hru
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88 Beaty et al. 34. Peters, D. G.,
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5 Molecular Classification of Breas
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Molecular Profiling of Breast Cance
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Fig. 2. (Continued) the right ident
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6 Discovery of Differentially Expre
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Gene Target Discovery 117 In the fi
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Gene Target Discovery 119 sequences
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Gene Target Discovery 121 There is
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Gene Target Discovery 123 digestion
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Gene Target Discovery 125 Fig. 1. P
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Gene Target Discovery 127 5. Sargen
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Gene Target Discovery 129 41. Berry
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132 Matsumoto, Miyagishi, and Taira
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144 Fig. 1. The ribozyme-expression
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146 Sano and Taira 5. 10X L (low-sa
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148 Sano and Taira ribozymes may cl
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150 Fig. 3. A system for the identi
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152 Sano and Taira 4. Notes 1. We r
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9 Production of siRNA- and cDNA-Tra
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Transfected Cell Arrays 157 Fig. 1.
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Transfected Cell Arrays 159 2. The
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Transfected Cell Arrays 161 5. Simp
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164 Houdebine Fig. 1. Different met
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166 Houdebine concentrations become
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168 Houdebine Fig. 2. Major methods
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170 Houdebine integrate into the me
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172 Houdebine 2.2.3. Knock-In Into
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174 Houdebine as insulators. The co
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176 Houdebine Fig. 5. Different met
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178 Houdebine systems. Moreover, st
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180 Houdebine contribute to generat
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182 Houdebine Fig.7. Example of a v
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184 Houdebine more extensively. Tra
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186 Houdebine and stroma. Several o
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188 Houdebine explained at the mole
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190 Houdebine of the intestinal epi
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192 Houdebine interference of the g
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194 Houdebine 17. Whitelaw, C. B. A
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196 Houdebine 51. Bell, A. C., West
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198 Houdebine 86. Carmichael, G. G.
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200 Houdebine 121. Pailhoux, E., Vi
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202 Houdebine 156. Auerbach, A. B.,
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204 Vijayaraj, Söhl, and Magin 1.
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206 Vijayaraj, Söhl, and Magin Fig
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208 Vijayaraj, Söhl, and Magin fil
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Page 266 and 267: 12 The HUVEC/Matrigel Assay An In V
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Overview of Immune System 311 Fig.
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Overview of Immune System 313 measu
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Overview of Immune System 315 42. H
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Overview of Immune System 317 74. D
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15 Potential Target Antigens for Im
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Tumor Antigen Discovery 321 develop
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Tumor Antigen Discovery 323 panel b
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Tumor Antigen Discovery 325 16. Joc
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16 Identification of Tumor Antigens
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Immunoproteomics 329 by “shot gun
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Immunoproteomics 331 isoelectric fo
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Immunoproteomics 333 3. 2D-PAGE is
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17 Protein Arrays A Versatile Toolb
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Protein Arrays 337 Table 1 Classifi
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Protein Arrays 339 fractions from c
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Protein Arrays 341 combinatorial sc
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Protein Arrays 343 The ideal proteo
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Protein Arrays 345 18. Cekaite, H.
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Protein Arrays 347 49. Yuko Kawahas
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Index 349 Index A Acetyl-CoA carbox
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Index 351 conditional by using FRT
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Index 353 Recombinant tumor antigen
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